In the present study, we compared mesenchymal stem cells (MSCs) derived from 4 different sources, human bone marrow (BM), adipose tissue (AT), umbilical cord Wharton’s Jelly (WJ) and the placenta (PL), in order to determine which population of MSCs displayed the most prominent immunosuppressive effects on phytohemagglutinin-induced T cell proliferation, and which one had the highest proliferative and differentiation potential. MSC and T lymphocyte co-culture (mixed culture) was used to determine whether the MSCs inhibit T cell proliferation, as well as which population of MSCs has the strongest inhibitory ability. The expression of immune-related genes was analyzed by RT-PCR and RT-qPCR. The proliferation and differentiation potential of the MSCs were determined using standard methods. Following MSC and T cell co-culture, mitogen-induced T cell proliferation was effectively suppressed by all 4 populations of MSCs. This occurred through soluble factors rather than direct contact inhibition. Among the 4 populations of MSCs, the WJ-MSC has the strongest suppression effects. On immune related genes, WJ-MSC has the weakest expression of MHC II genes, TLR4, TLR3, JAG1, NOTCH2 and NOTCH3. To compare the proliferation potential, WJ-MSCs showed the most rapid growth rate followed by the AT-, PL- and BM-MSCs. As regards differentiation potential, the WJ-MSCs had the strongest osteogenetic ability followed by PL, AT and BM-MSC. AT-MSC has the strongest adipogenetic ability followed by the WJ-, BM- and PL-MSCs. These data indicated that the WJ-MSCs had the strongest immunomodulatory and immunosuppressive potential. In light of these observations, we suggest that WJ-MSCs are the most attractive cell population for use in immune cellular therapy when immunosuppressive action is required. In the early 1970s, Friedenstein teocytes . Subsequteocytes ,4. MSCs teocytes . Among tteocytes ,7.In vitro and in vivo studies have also demonstrated that MSCs can differentiate into cells of non-mesodermal origin, such as neurons, skin and gut epithelial cells, hepatocytes and pneumocytes  for 3 min at 37°C. The cells were washed and harvested by centrifugation at 1000 rpm for 5 min, then replanting at a lower density .MSCs were isolated from 4 different sources: BM, AT, WJ and PL tissue. The BM and AT samples were obtained from healthy volunteer donors, while the WJ and PL samples were from tissue following normal caesarean birth. All individuals provided written informed consent and the study was approved by the Ethics Committee of the China-Japan Union Hospital, Jilin University, Changchun, China. The age range of the donors was as follows: the BM was from individuals aged 18 to 43 years, the AT was from individuals aged 23 to 50 years, and the WJ and PL tissue were from individuals aged 23 to 38 years. The MSCs derived from BM, AT, WJ and PL were isolated according to previously described methods –27 with The MSCs were removed from the culture flasks by incubation in 0.05% trypsin-EDTA at 37°C and then washed twice with PBS. The cell suspensions were then incubated with antibodies against CD44-phycoerythrin (PE), CD73-PE, CD14-PE, CD34-PE, CD90-fluorescein isothiocyanate (FITC), CD45-FITC and CD105-PerCP  and protected from light for 30 min at 4°C. Following incubation, the cells were washed twice with PBS. The fluorescence intensity of the cells was evaluated using a flow cytometer  and the data were analyzed using CellQuest software (BD Biosciences).4 MSCs/cm2 was seeded into a 12-well plate and incubated in MSC growth medium (α-MEM with 10% FBS) at 37°C in a humidified atmosphere with 5% CO2 for a minimum of 2 h or up to 4 days (to near or complete confluence) before re-seeding with adipogenic differentiation medium . The MSCs will continue to undergo limited expansion as they differentiate under adipogenic conditions. The cultures were re-fed every 3–4 days. After 21 days under differentiating conditions, the medium was removed from the wells, and the cells were rinsed once with PBS, and fixed with 4% formaldehyde solution for 30 min. After fixation, the cells were rinsed twice with PBS, stained with Oil Red O (Sigma) for 15 min, rinsed twice with PBS and visualized under a light microscope . Images were captured for qualitative and quantitative analyses. Fifteen fields at x400 magnification under the microscope were randomly selected and the stained cell and total cell numbers were counted. The sample number was 3 each for all 4 populations of MSCs.A total of 5x104 cells/cm2. The cells were fed in α-MEM with 10% FBS and incubated at 37°C in a humidified atmosphere with 5% CO2. Osteogenic differentiation medium  was added to the cells the second day after the cells attached to the bottom of the petri dish. MSCs will undergo limited expansion during differentiation under osteogenic conditions. The cultures were re-fed every 3–4 days. On day 35, the medium was removed from the 12-well plate and the cells were rinsed twice with PBS. The cells were fixed with 4% formaldehyde solution for 30 min. After fixation, the cells were rinsed with distilled water and stained with 2% Alizarin Red S solution  for 5 min. The cells were rinsed 3 times with distilled water and visualized under a light microscope (Olympus). Images were captured for qualitative or quantitative analyses. Bone nodule numbers of a total of 3 wells were counted for each sample. Three samples from each population of MSCs were analyzed.The MSCs were seeded into a 12-well plate at 5x105/well. Each population of MSCs was seeded in triplicate. Every 4 days, the cells were harvested with trypsin/EDTA and counted using a hemacytometer for up to 8 passages. The mean value of the cell number counts was calculated from 7 tissue samples from each cell population and the mean population doubling time was obtained for each passage according to the following formula: population doubling time = T x lg2/(lgNt - lgN0), where T is the culture time, N0 is the initial cell number and Nt is the harvested cell number.The 4 populations of MSCs beginning from passage 1 were trypsinized from the culture discs before plating into the 6-well plates at a density of 10Total cellular RNA was extracted using TRIzol reagent  according to the manufacturer’s instructions. RNA integrity was electrophoretically verified by ethidium bromide staining and a OD260/OD280 nm absorption ratio (OD >1.9). Total RNA (500 ng) was reverse transcribed using AWV reverse trancriptase, oligo(dT)15  according to the manufacturer’s instructions. No amplification control (NAC) was included without adding reverse transcriptase. Reactions were incubated at 42°C for 30 min, then 95°C for 5 min and 5°C for 5 min.Twenty five nanograms of cDNA was used in a 25-μl reaction volume with 0.15 μl Takara Ex Taq HS (Takara), 5 μM of gene-specific forward and reverse primers , 5 μl 5X PCR buffer and sterile water. Reactions were incubated at 94°C for 30 sec, 56°C for 30 sec and 72°C for 30 sec for total 30 cycles.http://flypush.imgen.bcm.tmc.edu/primer/primer3_www.cgi) and synthesized with the temperature at 60°C  were added to inhibit MSC proliferation, and the cells were incubated for 2 h at 37°C followed by 5 extensive washes with medium. A total of 105 T cells/well was added and stimulated with 10 g/ml phytohemagglutinin  and 10 ng/ml interleukin (IL)-2 (Sigma). IL-2/PHA-activated T cells were cultured in the presence or absence of MSCs. The cultures were plated in triplicate and incubated for 5 days before the addition of 5-bromo-20-deoxyuridine (BrdU). After 18 h, proliferation was assessed using the BrdU-Assay kit  according to the manufacturer’s instructions.The co-culture of the MSCs and T cells was carried out using 24-well plates (contact culture) and the Transwell system (isolated culture) in which the T cells and MSCs were physically separated by a membrane permeable for soluble factors. The inner hanging cell culture insets with 0.4-μm pore size membrane of a 24-well plate were purchased from Millipore . The BM-, AT-, WJ- and PL-MSCs were seeded at 10Statistical analyses were performed using SPSS software version 17.0 . The differences among the treatment groups were analyzed by one-way analysis of variance (ANOVA) and two-way ANOVA with post-hoc analysis using the Dunnett’s test. Parametric data are expressed as the means ± standard deviation (SD). A value of P<0.05 was considered to indicate a statistically significant difference.The MSCs derived from 4 different sources  presented the typical morphology of fibroblastic cells and displayed a high capacity to adhere to the plastic disc . FollowiThe cell-surface antigen profiles of these cells after 3 passages in culture were analyzed by flow cytometry. These cells were strongly positive for MSC-specific surface markers, such as CD44, CD73, CD90 and CD105, but negative for CD14, CD34 and CD45 . As regaOil Red O staining revealed that the MSCs were positive for lipid vesicle-forming adipocytes , and calAs regards osteogenesis, the cell volume began to increase after 1 week of induction. The shape of the cells gradually became triangular or polygonal and granular matter appeared in the cytoplasm. After 3 weeks of induction, yellow substances accumulated in the MSCs from all 4 tissue sources, particulary in the WJ-MSCs, and gradually, these sediments increased in size to form round or polygonal nodules . A totalDuring cell proliferation, the MSCs were cultured up to passage 8. The WT-MSCs displayed the highest cumulative cell population followed by the AT-, PL- and BM-MSCs . Based oThe MSCs were screened for their surface expression of HLA antigens, co-stimulatory factors and immune tolerance molecules. Through RT-qPCR of HLA-DMA, HLA-DPB1 and HLA-DRA, we found that the BM-MSCs had the highest expression of MHCII molecules than the other MSC populations. The WJ-MSCs had the lowest expression of these molecules among the 4 cell populations. The expression of DMA, DRA and DPB1 in the BM-MSCs was 16-, 36- and 4-fold higher, respectively compared with the WJ-MSCs . The immPHA/IL-2-activated T cells were incubated on a layer of MSCs derived from BM, AT, WJ and PL tissue. T cell proliferation decreased significantly following co-culture with the MSCs. The ratio of growth retardation of the T lymphocytes in the contact culture was: BM-MSCs, 41.4±3.2%; AT-MSCs, 35.5±1.7%; WJ-MSCs, 22.6±0.9% and PL-MSCs, 30.4±1.2% when compared to the T cells cultured alone. The results from Transwell culture were similar to those of the contact culture (P>0.05). The inhibitory effects of the AT-MSCs, WJ-MSCs and PL-MSCs on T cell proliferation were more prominent than those of the BM-MSCs (P<0.01). The inhibitory effects of the WJ-MSCs on T cell proliferation were the most prominent P<0.01; .et al also revealed that matrix cells from the WJ showed similar characteristics with those of MSCs derived from BM (BM is the earliest tissue source for MSC extraction and represents the most commonly used source for MSCs in clinical treatments. However, there are some limitations in using BM-MSCs due to the high degree of viral exposure and the significant decrease in cell number and proliferation/differentiation capacity with the increasing age of the donor ,30. It i from BM . In addt from BM . Human BIn this study, we performed a side-by-side comparison of 4 populations of MSCs derived from BM, AT, WJ and PL simultaneously. We observed significant differences in the proliferative potential among the 4 populations of MSCs; the WJ-MSCs exhibited the highest growth rate. The growth curve showed that the proliferative capacity of the WJ-, AT- and PL-MSCs was significantly greater than that of the BM-MSCs. These data coincide with those of previous studies –37.Some believe that the growth differences may not reflect compartment-specific characteristics, but most probably reflect differences in newborn and adult donors . AlternaMSCs derived from different tissues have been demonstrated in a large number of studies to differentiate into cells in the mesodermal lineages, such as osteoblasts and adipocytes –44. Our et al (As regards osteogenesis, the results revealed that the WJ-MSCs had the greatest potential in osteogenesis followed by the PL-MSCs, AT-MSCs and MB-MSCs. Since the WJ and PL tisssues were obtained from individuals who were much younger than the individuals the AT and BM tissues were obtained from, we predicted that the MSCs derived from younger tissue sources would differentiate more easily into osteoblasts that those obtained from older tissue sources. Baksh et al  also demIt is a widely accepted fact that MSCs have immunosuppressive and immunomodulatory functions. Due to their ability to regulate immune responses, MSCs are a potential candidate for treating a wide range of immune-mediated diseases ,46. TherHLA-DRA is one of the HLA class II α chain paralogues. This class II molecule is a heterodimer consisting of an α and a β chain, both anchored in the membrane. It plays a central role in the immune system by presenting peptides derived from extracellular proteins, such as HLA-DPB1. HLA-DMA is an antigen processing and presentation-related gene. Our data demonstrated that the WJ-MSCs expressed the lowest levels of the HLA class II genes, HLA-DRA, HLA-DPB1 and HLA-DMA, than any other 3 populatinos of MSCs, while the BM-MSCs expressed the highest level of these 3 genes. These data suggest that the WJ-MSCs may be the most effective population of MSCs for clinical application.We also analyzed the mRNA expression of JAG1, TLR4, TLR3, NOTCH2 and NOTCH3 since they are expressed in MSCs and are closely related to immune response . Our datin vitro (in vivo (It has been shown that MSCs possess the intrinsic homing ability to migrate to injured tissues and actively participate in tissue repair. In addition, MSCs possess the unique ability to suppress immune responses, both in vitro  and in v(in vivo . In orde(in vivo . Our dat(in vivo . Studies(in vivo . Other s(in vivo . To deteIn this study, we demonstrate that the proliferative potential of WJ-MSCs is higher than that of AT-MSCs, PL-MSCs and BM-MSCs. WJ-MSCs have the strongest osteogenic ability followed by the PL-MSCs, AT-MSCs and BM-MSCs. WJ-MSCs have the weakest expression of MHC II genes, while BM-MSC have the highest. WJ-MSCs have the weakest expression of the immune-related genes, TLR4, TLR3, JAG1, NOTCH2 and NOTCH3. Furthermore, WJ-MSCs have the most prominent suppressive effect on T lymphocytes among the 4 populations of MSCs in the co-culture experiments. Therefore, WJ-MSCs may be one of the best sources of MSCs for tissue regeneration in future clinical application.


Coreopsis tinctoria Nutt. flower extracts on hyperglycemia of diet-induced obese mice and the underlying mechanisms. Coreopsis tinctoria flower was extracted with ethanol and water, respectively. The total phenol, flavonoid levels, and the constituents of the extracts were measured. For the animal experiments, C57BL/6 mice were fed with a chow diet, high-fat diet, or high-fat diet mixed with 0.4% (w/w) water and ethanol extracts of Coreopsis tinctoria flower for 8 weeks. The inhibitory effects of the extracts on α-glucosidase activity and the antioxidant properties were assayed in vitro. We found that the extracts blocked the increase of fasting blood glucose, serum triglyceride (TG), insulin, leptin, and liver lipid levels and prevented the development of glucose tolerance impairment and insulin resistance in the C57BL/6 mice induced by a high-fat diet. The extracts inhibited α-glycosidase activity and increased oxidant activity in vitro. In conclusion, Coreopsis tinctoria flower extracts may ameliorate high-fat diet-induced hyperglycemia and insulin resistance. The underling mechanism may be via the inhibition of α-glucosidase activity. Our data indicate that Coreopsis tinctoria flower could be used as a beverage supplement and a potential source of drugs for treatment of diabetics.The aim of this study was to assay the effects of Next, 40 μL of reaction mixtures was placed into 96-well plates, and 160 μL of 0.1 M sodium carbonate solution was added to terminate the reaction. Absorbance was recorded at 400 nm using microplate reader. Acarbose was used as the positive control. The α-glycosidase inhibitory activity is expressed as the inhibition percentage and was calculated as follows: inhibition (%) = (1 − ΔAsam/ΔAcon) × 100%. Δ was defined as absorbance of the sample or the control.The ications . A mixtuThe free radical-scavenging activities of EEC and WEC were assayed by using kits from Biyuntian  for 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity, 2,2′-azino-bis 3-ethylbenzothiazoline-6-sulphonic acid (ABTS) radical-scavenging activity, and Ferric-reducing ability power (FRAP) according to our previous report .P < 0.05.The data are expressed as mean ± SD. Data analyses of the control and the prevented groups were performed using the SPSS 21.0 for Windows statistical program. Differences were regarded as statistically significant when We characterized the constituents of the extracts using LC-MS. Female C57BL/6 mice were fed with a chow diet (Chow) or a high-fat (HF) diet or a HF diet mixed with 0.4% EEC and WEC, respectively, for 8 weeks. At the end of the treatment, the body weight of the HF control was significantly higher than that of the chow diet-fed mice. Although the food intake in the EEC and WEC groups was lower than that in the HF group , the bodP < 0.05), while the fasting blood glucose levels were lower in EEC- and WEC-treated groups compared to that in the HF group  assay. The radical-scavenging activities of WEC and EEC had DPPH ICectively . The actectively . As showectively , the tot Coreopsis tinctoria flower is used in beverages because of its flavor and its potential health benefits. In the present study, we verified that the extracts of Coreopsis tinctoria flower could lower blood glucose levels through the inhibition of α-glycosidase activity and via its antioxidant properties.The driedPrevious studies have shown that CTF is rich in flavonoids and polyphenols , 18. SimCoreopsis tinctoria flower has traditionally been used to control diabetes in Portugal [ Coreopsis tinctoria flower infusion reduced blood glucose levels in glucose-intolerant Wistar rats [ Coreopsis tinctoria flower is probably through promoting the impaired pancreatic cells.Portugal , 9. It htar rats  and imprtar rats  and gluctar rats . This fu Coreopsis tinctoria flower may prevent the increase of blood glucose levels and the development of insulin resistance in mice induced by a HF diet. This effect was further supported by the data of serum insulin, adiponectin, and leptin levels in the DIO mice, which were reversed by the treatment of CTF extracts. This diabetic model is caused by insufficient insulin sensitivity [In the present study, we identified that the extracts ofsitivity , but notα-Glucosidase is an intestinal enzyme that digests carbohydrates to release glucose [α-glucosidase may suppress the rate of digestion of carbohydrates and block the absorption of glucose from food [α-glucosidase inhibitors may decrease blood glycated hemoglobin A1C level [α-glycosidase, such as acarbose [α-glucosidase activities in vitro that were similar to acarbose (α-glucosidase inhibitor. We assumed that the active compound is flavonoid, because only about 1% of flavonoid can be absorbed in intestine. However, which constituent is involved in the α-glucosidase inhibitory effect remains uncertain because it is difficult to obtain the compounds. glucose . Inhibitrom food , 22. In 1C level , 24. Inhacarbose –27, haveacarbose , suggestα-glucosidase may improve lipid disorders. However, we cannot exclude that other mechanisms may be involved in the action.We also observed that the EEC prevented the increase of serum triglyceride contents in DIO mice. It has been reported that the administration of acarbose also improves postprandial hyperlipidemia , 28. Lon Coreopsis tinctoria flower displayed antioxidant activities [As abundant evidence has implied, oxidative stress is associated with a wide range of diseases , 7. Antitivities , 32. In  Coreopsis tinctoria flower were shown to prevent the increase of blood glucose levels and glucose tolerance impairment of mice induced by a high-fat diet. The hypoglycemic effects of the extracts may be exerted through the inhibition of α-glucosidase activity and the antioxidant properties. Our study provides evidence for the use of Coreopsis tinctoria flower to treat or prevent diabetes through the multiple mechanisms and suggests that the Coreopsis tinctoria flower may be used as a nutriceuticals or potential source of drug for diabetes.In conclusion, the extracts of the


We applied this approach to study the metabolic dynamics in amino acid perturbation of Saccharomyces cerevisiae. In metabolic turnover analysis, 69 peaks including 35 unidentified peaks were investigated. Multivariate analysis of metabolic turnover successfully detected a pathway known to be inhibited by amino acid perturbation. In addition, our strategy enabled identification of unknown peaks putatively related to the perturbation.Isotope-labeling is a useful technique for understanding cellular metabolism. Recent advances in metabolomics have extended the capability of isotope-assisted studies to reveal global metabolism. For instance, isotope-assisted metabolomics technology has enabled the mapping of a global metabolic network, estimation of flux at branch points of metabolic pathways, and assignment of elemental formulas to unknown metabolites. Furthermore, some data processing tools have been developed to apply these techniques to a non-targeted approach, which plays an important role in revealing unknown or unexpected metabolism. However, data collection and integration strategies for non-targeted isotope-assisted metabolomics have not been established. Therefore, a systematic approach is proposed to elucidate metabolic dynamics without targeting pathways by means of time-resolved isotope tracking,  Isotope-labeling experimentation is a useful tool for understanding cellular metabolism. Tracking the fate of isotope-labeled substrates has played an important role in determination of metabolic pathways such as the Calvin-Benson cycle  and the 13CMS [Recent advances in metabolomics enabled monitoring of a huge number of metabolites with high reproducibility . Metabol13CMS ,14,15. S13CMS ,17,18, tet al. introduced non-targeted isotope-assisted metabolomics to elucidate perturbation-related pathways using X13CMS [i.e., “metabolic turnover analysis,” could overcome this problem [Recently, Huang g X13CMS . This cog X13CMS , snapsho problem ,21,22, tSaccharomyces cerevisiae.Here we suggest a systematic approach for elucidation of the unknown and unexpected pathways related to perturbation by means of non-targeted metabolic turnover analysis and multivariate analysis . This ap13C-labeled samples were also prepared to facilitate peak picking. All detectable metabolites were monitored using scan-mode analysis on a mass spectrometer. Processes III and IV present the methods for data analysis including peak picking, isotope ratio calculation, and multivariate analysis. In Process III, the peaks containing carbon atoms are selected from the data set of both non-labeled and 13C-labeled samples. Then, the metabolic turnover of each peak was calculated. In Process IV, differential analysis was performed to elucidate perturbation-related pathways and rate the priority of the peaks. High priority peaks were annotated based on several aspects. Finally, the effect of biological perturbation is discussed in Process V.The scheme for non-targeted metabolic turnover analysis is shown in 13C-labeled samples was used  of each metabolite was calculated using the following equation:PA represents peak area, t represents each sampling time, k represents the m/z of a fragment of the metabolite, min represents the m/z of 12C-fragments, and max represents 13C-fragments of the metabolite described on m/z of the 12C-monoisotope to the 13C-monoisotope of a fragment ion. Only the IRt,min) of each metabolite was calculated by subtracting the average at each time point as follows:s represents each sample, t represents the sampling time, and n represents the sample number including all sample types at time t. The DIR was then applied to PCA, taking every sampling time for each sample as an independent class and DIR as the element. Since the differences among the DIRs of the samples were important, preprocessing was not performed.Differential analysis . Therefot-test can be applied to analyze several replicates; however, the appropriate time point for differential analysis is difficult to estimate since the isotopically non-stationary phase depends on the metabolites, labeling sources, conditions, and organisms employed. In addition, a t-test of successive data requires a data integration process for effective detection and an estimation of appropriate data points to avoid multiple comparison problems. The Friedman test is another alternative for the analysis of successive data. However, its sensitivity also depends on the sampling time and number because the turnover data has a nonparametric and standardized data structure. Particularly, the sampling number of the isotopically stationary phase probably affects to the detection power because the DIR approaches zero at those points. To avoid these problems, we used PCA in this study.Statistical tests provide alternative analyses with the following considerations: (i) the sampling point; (ii) the sampling number; and (iii) the analysis of successive data. The two-sample DIR variance, and the second was calculated to orthogonally maximize the sample differences to the first coordinate. Since the DIR approaches zero at early time points and at late, isotopically stationary time points, isotopically non-stationary time points are scattered from the origin on the score plot  and n represent the factor loading and the sample number, respectively. The test statistic, test, has a t-distribution with (n − 2) degrees of freedom.Candidate metabolites from fluctuated pathways were selected using the PCA loading plot b. Recentloadings ; separatThe resulting PCA score plot displays the different sample types and sampling times a, and thThe selected peaks were identified automatically . Of the 12C and 13C isotopomers of the detected fragments  [The ROSCARF) . The BY4Leucine is known to inhibit many reactions on the branched chain amino acid pathway , and ourTime courses of isotopomer ratios were analyzed in this study. On the other hand, the labeling patterns at isotopically stationary phase are often used to elucidate metabolic dynamics. The labeling pattern provides information about branches and confluences on the metabolic map. Therefore, the patterns are used to calculate the flux in central metabolic pathways, specifically glycolysis, the pentose phosphate pathway, and the TCA cycle ,13. HoweS. cerevisiae is relatively low under minimum medium, the detected peaks are limited [In this experiment, 69 peaks were detected as carbon containing peaks. In spite of non-target analysis, less than a hundred peaks were observed. This is probably caused by three reasons. Firstly, the intensity of the metabolites is not sufficient for the analysis. Since the metabolite abundance of  limited ; SecondlIn the present study, we analyzed a well-known perturbation and successfully detected the metabolic pathway known to be affected in a single measurement. However, multiple measurements or supplementary techniques will be required for the analysis perturbations with unknown effects to accurately and precisely detect candidate pathways. Since our approach may suggest ideas contrary to common knowledge, we recommend that conclusions be verified with established techniques.d-glucose was purchased from Nacalai Tesque . 13C6-d-glucose was purchased from Cambridge Isotope Laboratory . For metabolite extraction and analysis, HPLC-grade methanol, HPLC-grade chloroform, HPLC-grade distilled water, and pyridine were purchased from Wako . Methoxyamine hydrochloride was purchased from Sigma-Aldrich . N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSATFA) was purchased from GL Sciences .Yeast extract, peptone, and a yeast nitrogen base without amino acid were purchased from BD . Saccharomyces cerevisiae BY4742  and reference strain X2180  were used for the experiment. The strain from the glycerol stock was streaked onto a yeast extract peptone dextrose (YPD) agar plate  to obtain a single colony isolate. For metabolic turnover analysis, cells from a single colony were picked and pre-cultured by inoculation onto 5 mL of synthetic defined (SD) medium  with or without amino acid supplement  and incubated overnight at 30 °C. The culture was then inoculated to 50 mL of fresh medium of the same composition as the pre-culture to an OD590 of 0.01 and grown at 30 °C in a rotary shaker (200 rpm) to an OD590 of 1.5. For the 0 s time point, 5 mL of culture was collected using a plastic syringe and syringe filter . The syringe filter was immediately soaked in liquid nitrogen to quench metabolism. The other cells were harvested rapidly by vacuum filtration using a polytetrafluoroethylene (PTFE) membrane filter . The filter was immediately placed onto 50 mL of fresh medium to re-suspend the cells. The medium composition was the same as the pre-culture except that d-glucose was replaced with 10 g/L U-13C6-d-glucose. The cells were incubated at 30 °C, and 5 mL of culture was collected at 10, 20, 40, 80, 160, 320, 640, 1280, and 2560 s after the suspension point using a syringe filter with the procedure described above. The samples were freeze-dried and stored at −80 °C until extraction.12C-peaks and 13C-peaks, cells of the X2180 strain from a single colony were inoculated to 5 mL of SD medium containing 12C-glucose or 13C-glucose and incubated overnight at 30 °C as a pre-culture. The culture was inoculated to 50 mL of fresh medium of the same composition as the pre-culture to an OD590 of 0.01 and grown at 30 °C in a rotary shaker (200 rpm) to an OD590 of 1.5. The cells were then collected by centrifugation , freeze-dried, and stored at −80 °C until extraction.For reference samples of g, 4 °C, 5 min), and the supernatant was collected in a 1.5-mL plastic tube. The sample was centrifugally dried and freeze-dried. The pellet was stored at −80 °C until GC/MS analysis.For metabolic turnover analysis, 1 mL of mixed solvent (chloroform:methanol:water = 2 : 5 : 2) was applied to a syringe filter containing the yeast cells. The filtrate was collected and reapplied to the same filter. This procedure was performed two times. Then, 500 μL of distilled water was added to the filtrate and the mixture was vortexed. The sample was centrifuged  was added before incubation at 30 °C for 90 min. For trimethyl silylation, 50 μL of MSTFA was added before incubation at 37 °C for 30 min.m/z.GC/MS analysis was performed using a GC-2010 Plus gas chromatograph (Shimadzu) with an AOC-20is series injector/autosampler  and a GCMS-QP2010 Ultra mass spectrometer (Shimadzu). A 30 m long × 0.25 mm inter diameter fused silica capillary column coated with 0.25 μm CP-SIL 8 CB low bleed/MS  was used. The front inlet temperature was 230 °C. The helium gas flow rate through the column was 1.12 mL/min. The column temperature was held isothermally at 80 °C for 2 min and then ramped from 80 to 330 °C by 15 °C/min and held isothermally for 6 min. The transfer line and ion-source temperatures were 250 and 200 °C, respectively. Twenty scans per second were recorded over the mass range 85–500 13C-labeled reference samples using GCMSsolution 4.11 (Shimadzu). The mass spectra of co-eluting peaks in both references were compared. The peaks were assigned as carbon containing peaks when the topologies of these two spectra were similar with higher m/z of some fragments in the 13C-labeled sample  was calculated using Equation (1). The data were standardized by the maximum value of each metabolite.For metabolic turnover analysis, peaks and fragment ions labeled by carbons were manually selected by comparing data from non-labeled and le e.g., b. The shMetAlign  and AIouSIMCA 13  was used for principal component analysis (PCA). For differential analysis, values were calculated using Equation (2).To achieve an unbiased understanding of metabolic dynamics, a non-targeted approach to isotope-assisted metabolomics is necessary. Time-resolved monitoring of isotopomers is essential for the analysis of global metabolism because the isotope labeling speed differs in a metabolite-dependent manner. Herein, we developed a framework for a non-targeted approach to metabolic turnover analysis using GC/MS. The combination of metabolic turnover analysis and multivariate differential analysis efficiently visualized the important perturbation-related peaks. By combining isotope and turnover data, metabolites were informatively annotated. In the present study, we focused on GC/MS analysis; however, this approach can be applied to the other analytical platforms such as liquid chromatography-MS and capillary electrophoresis-MS. We expect that this approach will provide new insight into perturbation-related metabolism, especially for chemicals, genes, and diseases that are biologically not well understood.


Saccharomyces cerevisiae and non-Saccharomyces yeast strains is emerging for production of wines with improved complexity of flavor. The rational use of this approach is, however, dependent on knowing the impact that co-inoculation has in the physiology of S. cerevisiae. In this work the transcriptome of S.cerevisiae was monitored throughout a wine fermentation, carried out in single culture or in a consortium with Hanseniasporaguilliermondii, this being the first time that this relevant yeast–yeast interaction is examined at a genomic scale.The introduction of yeast starter cultures consisting in a blend of H. guilliermondii reduced the overall genome-wide transcriptional response of S. cerevisiae throughout the fermentation, which was attributable to a lower fermentative activity of S. cerevisiae while in the mixed-fermentation. Approximately 350 genes S. cerevisiae genes were found to be differently expressed (FDR < 0.05) in response to the presence of H. guilliermondii in the fermentation medium. Genes involved in biosynthesis of vitamins were enriched among those up-regulated in the mixed-culture fermentation, while genes related with the uptake and biosynthesis of amino acids were enriched among those more expressed in the single-culture. The differences in the aromatic profiles of wines obtained in the single and in the mixed-fermentations correlated with the differential expression of S. cerevisiae genes encoding enzymes required for formation of aroma compounds.Co-inoculation with S. cerevisiae to the challenging environment of mixed culture fermentation. The availability of nutrients, in particular, of nitrogen and vitamins, stands out as a factor that may determine population dynamics, fermentative activity and by-product formation.By integrating results obtained in the transcriptomic analysis performed with physiological data our study provided, for the first time, an integrated view into the adaptive responses of The online version of this article (doi:10.1186/s12934-015-0318-1) contains supplementary material, which is available to authorized users. Saccharomyces yeasts have been examined as potential adjuncts to Saccharomyces cerevisiae exploiting their flavor properties in order to respond to the new challenges of consumer demands for wines with high complexity of flavor and stylistic distinction [Saccharomyces yeasts on wine composition has been found to be influenced by the species/strains of Saccharomyces and non-Saccharomyces used; by the size of the inocula and by the timing of inoculation , among other factors [reviewed in Saccharomyces yeasts have also been found to have an inhibitory effect over S. cerevisiae growth, presumably due to the production of toxic compounds such as fatty acids and killer factor [S. cerevisiae strains when co-cultured with non-Saccharomyces species [Various non-tinction –5. This r factor –10. In a species , 11, 12. species .S. cerevisiae as an experimental system in life sciences research [S. cerevisiae during wine fermentation at different genomic levels including transcriptome, proteome and metabolome [S. cerevisiae wine yeasts [S. cerevisiae physiology, has provided valuable data of practical interest for the control and prevention of slow and premature fermentation arrest during winemaking and for the clarification of the impact of nitrogen metabolism of S. cerevisiae on aroma compounds formation during alcoholic fermentations. OMICS analyses also have the potential to provide a clear cut picture of the molecular mechanisms by which S. cerevisiae responds to the presence of other microbes in the environment, however, up to now only a few studies have addressed that issue in the context of wine fermentations. Recently, the transcriptome-wide response of yeast cells in mixed cultures with different wine bacteria had been elucidated including Lactobacillus delbrueckii subsp. bulgaricus, which co-occur with yeast in kefir fermentations [Oenococcus oeni, used for malolactic fermentation [S. cerevisiae-O. oeni interaction during winemaking involves not only indirect competition for nutrients, but also direct antagonistic responses. Although yeast–yeast interactions have not been examined at a genome-wide scale, some attempts have been made to examine the influence exerted by the presence of Starmerella bombicola, on the expression of a few selected S. cerevisiae genes [Saccharomyces species was found to lead to alterations in both expression and enzymatic activity of S. cerevisiae alcohol dehydrogenase 1 (encoded by ADH1 gene) and pyruvate decarboxylase (encoded by PDC1).Wine research has benefited enormously from the privileged position of research . The budresearch , which ptabolome –22. In pe yeasts , 23, 24.ntations  and Oenoentation –28, 29. ae genes . ExposurS. cerevisiae adjusts its transcriptome along fermentation of a natural grape must in single culture or in consortium with Hanseniasporaguilliermondii. To our knowledge this is the first study focused on the elucidation at the molecular level of this yeast–yeast interaction, a knowledge that could be used to guide the rational development of mixed blends composed by these two yeasts and of its subsequent utilization in mixed fermentations.In this study it was performed the first genome-wide analysis of how H. guilliermondii in consortium with S. cerevisiae [H. guilliermondii negatively affects S. cerevisiae growth and fermentation rate, irrespective of the initial nitrogen concentration of the grape-juice. Co-inoculation of S. cerevisiae with H. guilliermondii has also been found to significantly alter the panoply of aroma compounds found at the end of the fermentation [S. cerevisiae along a mixed wine-fermentation with H. guilliermondii were monitored using DNA microarrays. Since H. guilliermondii is a non-standard model yeast for which comprehensive DNA microarrays are not available, we have focused on the effect of the co-inoculation only in the alteration of the S. cerevisiae transcriptome. The experimental conditions used were the same as those described in [In a previous work the effect of grape-juice nitrogen availability on wine yeast mixed-culture fermentations has been evaluated using a strain of revisiae . The resentation . In thisribed in , being oribed in .H. guilliermondii impacted the transcriptome of S. cerevisiae throughout the fermentation, the data obtained from the microarrays experiments were subjected to Principal Component Analysis (PCA). This multivariate statistical analysis revealed that gene expression differences between the fermentation stages were much greater than those observed between the two inoculum types . Although the mean expression value of each gene along the fermentation is merely an arbitrary reference point, such way of analyzing gene expression mitigates the influence exerted by fermentation dynamics, while maintaining the aptitude to identify expression differences [S. cerevisiae transcriptome adjusts to the different dynamics of the single or mixed-culture fermentation; an information that would be missed if only cross-comparisons between expression levels in single vs mixed cultures had been performed. Only genes having an increased or decreased expression of at least twofold were considered to be up- or down- regulated in a given fermentation stage. Using this criterion, two sets of 2224 genes and 1406 S. cerevisiae genes were considered to be differently expressed along the single- or mixed-fermentations, respectively  analysis was performed considering all samples of the single-culture fermentation as a group and those of mixed-culture fermentation as another group, irrespective of the fermentation stage. This unsupervised approach led to the identification of 120 S. cerevisiae genes that seem to respond to the presence of H. guilliermondii during the course of fermentation, 85 being up-regulated in the presence of the non-Saccharomyces species and 35 down-regulated. A list of the top 10 S. cerevisiae genes whose expression varied the most in the presence of H. guilliermondii is shown in Table In this section the expression of S. cerevisiae in the single-culture, compared to the mixed culture, was GAP1 (8.7-fold) and AGP1 (13.4-fold), encoding general amino acid carriers with broad substrate ranges, as well as PUT4 (4.8-fold), encoding a specific proline transporter. These three genes are under the nitrogen catabolite repression (NCR) and their higher expression in the single-culture might suggest an alleviation of this repressive effective. Consistent with this idea, the NCR-repressed MEP1 and MEP2 genes, encoding the specific permeases for ammonium assimilation, were also found to be more actively transcribed in the single-culture fermentation than in the mixed fermentation, 1.97- and 1.95- fold, respectively. Although amino acid consumption profile was not assessed in this study, high levels of ammonium were detected at 24 h in both fermentations , and THI21 (7.9-fold), whose expression is regulated in the dependence of thiamine availability [THI20 and THI21 in the mixed-culture suggest that S. cerevisiae and H. guilliermondii might be competing for thiamine which could lead to a depletion of this vitamin in the must.In the pair wise comparison performed at 24 h only 27 genes were found to be differentially expressed (FDR < 0.05) between the single and mixed-culture fermentations  included in the so-called Fermentation Stress Response (FSR) [S. cerevisiae in single-culture, as discussed above. As observed after 24 h of fermentation, several genes more expressed in single-culture are known to be under the NCR response, namely those encoding proteins required for the uptake and utilization of allantoin—DAL5 (52.7-fold more expressed in the single-culture), DAL4 (5.7-fold)-; proline—PUT1 (55.3-fold), PUT4 (12.3-fold), PUT2 (6.0-fold), -and urea—DUR3 (15.4-fold), DUR1,2 (7.7-fold), as well as the regulator of nitrogen catabolite repression DAL80 (11.9-fold). Altogether these observations reinforce the concept that in single-culture S. cerevisiae is sensing nitrogen limitation, this being confirmed, at least in part, by the lower levels of ammonium available registered at 48 h in the single-culture fermentation, compared to the levels registered in the mixed culture fermentation  . The incof H. guilliermondii. Interestingly, almost all the genes required for biosynthesis of the purine nucleotide monophosphate are more expressed in the mixed-culture fermentation: ADE1 (4.8-fold), ADE2 (4.3-fold), ADE4 (4.2-fold), ADE5,7 (5.2-fold), ADE12 (3.8-fold) and ADE17 (5.7-fold). It is possible that the higher expression of these genes in the mixed culture could result from the higher concentration of ammonium present in the growth medium since this has been found to exert a negative effect in the uptake of adenine [The most significantly overrepresented category among the genes that were found to be more expressed in the mixed-culture fermentation is “amino acid biosynthesis” Table , which i adenine . Indeed, adenine .SNO1 (14.2-fold) and SNZ3 (4.0-fold). Since at this point of the fermentation S. cerevisiae cells have ceased growth, both in the mixed-culture and in the single-culture fermentations  and BIO5 (3.3-fold)—and in the uptake of thiamine—PHO3 (10.1-fold)—supports this hypothesis, as these genes’ expression has been described to be regulated in the dependence of the concentration of these vitamins present in the growth medium.Higher mRNA levels of several stationary growth-phase associated genes  were alsS. cerevisiae genes was significantly altered in both fermentations, 71 genes being more expressed in mixed-culture and 143 in the single culture (Table BIO3 (3.1-fold) and BIO5 (2.5-fold) which are involved in biotin biosynthesis, reinforcing the suggestion that depletion of vitamins is one of the main consequences of mixed-culture fermentations. The dataset of genes found to be more expressed in the single culture at this fermentation stage was very broad in terms of physiological function, not being possible to identify significantly enriched functional classes.At the final fermentation stage analyzed, the expression of 214 S. cerevisiae was cultivated in the presence of H. guilliermondii [2S were more abundant in the wines that were only fermented by S. cerevisiae  is also in agreement with the higher levels of isobutanol and 2-methyl-1-butanol detected in the wines produced by the mixed-cultures , although with different catalytic efficiencies [ADH1 gene product is the major enzyme responsible for converting acetaldehyde to ethanol [ALD2-6 [ALD4 and ALD6 are the major contributors of acetate formation during wine fermentations [ALD3 and ALD5 seem to be glucose-repressed [The two final steps of alcoholic fermentation involves the decarboxylation of pyruvate, catalyzed by pyruvate decarboxylases (PDC), yielding acetaldehyde which in turn is reduced by the activity of several iso-enzymes of alcohol dehydrogenase (ADH) to ethanol. In entation . Also ficiencies . The cyt ethanol . Acetald [ALD2-6 , 63. It epressed .S. cerevisiae single-culture  pathway  and sulfide incorporation . Also, the results obtained in this study are not in agreement with the previous suggestion [Hway Fig. a. The eft growth . Indeed,ion Fig. . The higduction, –68. On tggestion  that corH. guilliermondii impacted molecular responses of a S. cerevisiae wine yeast strain during a wine fermentation. This genome-wide analysis detected a large set of S. cerevisiae genes differentially expressed as a result of the presence of H. guilliermondii in the must. Several changes that could be detected in the transcriptome of S. cerevisiae appear to result from a cellular response to changes in nutrient availability in the fermenting must attributable to H. guilliermondii metabolic activity. These observations are of paramount interest since it is well recognized the effect of nitrogen availability on yeast growth and fermentation kinetics and on the production of the major metabolites arising from sugar fermentation that establish the wine aroma profile. Indeed, the presence of H. guilliermondii dramatically influenced the expression patterns of various flavor-active compounds associated genes, which could underlie the differences obtained on the aroma profiles of the wines. These findings raise the question whether the impact of non-Saccharomyces strains on the sensorial profile of wines results from an additive production of aroma compounds and/or from influencing the metabolic behavior of the fermentative yeast S. cerevisiae through modulation of the must nutritional properties.In this study, a transcriptomics-based approach was used to examine how In sum, our study underline the importance of such a global approach for the study of yeast–yeast interactions shedding light on the molecular basis of yeast dynamics during wine fermentation. This new information will be useful for the rational development of mixed-starter cultures o be use in winemaking industry.H. guilliermondii, previously isolated in our laboratory from a fermenting grape-juice from Douro Region [S. cerevisiae UCD522 was supplied by the Enology Culture Collection, Department of Viticulture and Enology, University of California, Davis, USA.A strain of o Region , was selS. cerevisiae UCD522 and a natural H. guilliermondii strain were used to conduct alcoholic fermentation, of a natural grape-juice, either in single or mixed-culture. The initial pH of grape-juice was 3.26 and the concentration of sugars and nitrogen were 23.4 Brix and 387 mg YAN/L, respectively. Starter cultures of each strain were prepared by growing the yeast overnight in 100 mL-flasks, containing 50 mL of synthetic grape-juice medium with 267 mg YAN/L, supplied as DAP [6 CFU/mL. The fermentations were conducted in 500 mL-flasks filled to 2/3 of their volume fitted with a side-arm port sealed with a rubber septum to allow anaerobic sampling, and were maintained at 20 °C in an orbital shaker set at 120 rpm. Samples were collected daily for assessing fermentation and growth parameters and, at the end of fermentations, for chemical analysis. Growth and fermentation parameters as well as the final concentration of aroma compounds in the wines can be found in Lage et al. [Fermentation conditions are described in Lage et al. . Brieflyd as DAP . The flaGlucose, fructose, glycerol and ethanol extracellular levels in the samples collected at the time points selected for transcriptomic analysis were determined with commercial biochemical kits .Cell samples for DNA microarray analysis were obtained from both single- or mixed culture fermentations  at three different points: 24, 48 and 96 h after inoculation. Total RNA extraction was performed according to the hot phenol method. Concentration and purity was determined by spectrophotometry and integrity was confirmed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay . RNA was processed for use on Affymetrix  GeneChip Yeast Genome 2.0 Arrays, according to the manufacturer’s GeneChip 3′ IVT Express kit user manual. Briefly, 100 ng of total RNA containing spiked in Poly-A RNA controls was used in a reverse transcription reaction  to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in a 16 h in vitro transcription (IVT) reaction to generate aRNA . Size distribution of the aRNA and fragmented aRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay. 5 µg of fragmented aRNA was used in a 100-µl hybridization cocktail containing added hybridization controls. 80 µl of mixture was hybridized on arrays for 16 h at 45 °C. Standard post hybridization wash and double-stain protocols  were used on an Affymetrix GeneChip Fluidics Station 450. Arrays were scanned on an Affymetrix GeneChip scanner 3000 7G.http://www.dchip.org, Wong Lab, Harvard) applying a probe set mask file excluding all probes on the array representing Schizosaccharomyces pombe transcripts. The arrays were normalized to a baseline array with median CEL intensity by applying an Invariant Set Normalization Method [Scanned arrays were analyzed first with Affymetrix Expression Console software for quality control. Subsequent analysis was carried out with DNA-Chip Analyzer (dChip) 2010  following a strategy for genomic DNA hybridizations to GeneChips developed by Hammond et al. [As d et al. . CleanupS. cerevisiae transcriptome in the response to the presence of H. guilliermondii throughout the fermentation, different analysis were performed. The transcriptomic data were first analyzed using a principal component analysis, PCA method. PCA was applied as an exploratory data analysis method to visualize differences between the diverse data sets. Also EPCLUST online software (http://ep.ebi.ac.uk/EP/EPCLUST/) was used for cluster analysis by the K-means method. For the identification of differentially expressed genes, the data were analyzed using Rank Product (RP) [P < 0.05, 5 % FDR) was used to identify differentially expressed genes between single- and mixed-culture fermentations, either irrespective of fermentation stage or at each fermentation stage separately. Comparisons by RP analysis were done using single-culture fermentations as one experimental group and mixed-culture fermentations as another experimental group, in order to identify specific genes associated with the response of S. cerevisiae to the presence of H. guilliermondii. The gene lists were analyzed for enrichment of functional categories using the FunSpec interpreter [http://funspec.med.utoronto.ca.To assess the changes in uct (RP) , as impluct (RP) . RP, a nerpreter , availabhttp://www.ncbi.nlm.nih.gov/geo) under accession number GSE66521.The microarrays hybridization data are available at the Gene Expression Omnibus  PCR to amplify cDNA products reversely transcribed from mRNA (RT) . We analyzed the expression of some genes, -PCR reaction conditions were 5 min at 95 °C for initial denaturation and activation of the DNA polymerase, followed by 50 cycles of denaturation at 95 °C for 30 s, annealing at the appropriate temperature specific for each primer pair for 30 s and extension at 60 °C for 30 s. The StepOne software  was programmed to collect real time fluorescence data during the annealing and extension steps. Meltcurve analysis was performed after every qRT-PCR run to verify the specificity of the primers and to detect the presence (if any) of primer dimers. No-template controls were included for each primer pair. After RT-PCR, standard curves were plotted using the StepOne software . The standard curve for each gene was generated from serial dilutions of cDNA [(−1/slope). The expression of each gene determined by real-time qRT-PCR was normalized to the expression of the house-keeping gene, ACT1 . Relative quantification of the expression of each gene at each sampling point  for S. cerevisiae in single- or in mixed-culture was determined by the ΔΔCt method [S. cerevisiae by the lack of amplification observed in H. guilliermondii cDNA in single culture fermentation.Real-time qRT of cDNA . The cort method . The qRT


P < 0.05) between the groups. RT-PCR analysis revealed significant changes between groups in several genes implicated in a variety of disorders ranging from PTSD, anxiety, mood disorders, and substance dependence.Posttraumatic stress disorder (PTSD) is characterized by the occurrence of a traumatic event that is beyond the normal range of human experience. The future of PTSD treatment may specifically target the molecular mechanisms of PTSD. In the US, approximately 20% of adults report taking herbal products to treat medical illnesses. L-theanine is the amino acid in green tea primarily responsible for relaxation effects. No studies have evaluated the potential therapeutic properties of herbal medications on gene expression in PTSD. We evaluated gene expression in PTSD-induced changes in the amygdala and hippocampus of Sprague-Dawley rats. The rats were assigned to PTSD-stressed and nonstressed groups that received either saline, midazolam, L-theanine, or L-theanine + midazolam. Amygdala and hippocampus tissue samples were analyzed for changes in gene expression. One-way ANOVA was used to detect significant difference between groups in the amygdala and hippocampus. Of 88 genes examined, 17 had a large effect size greater than 0.138. Of these, 3 genes in the hippocampus and 5 genes in the amygdala were considered significant ( Posttraumatic stress disorder (PTSD) is characterized by the occurrence of a traumatic event that is beyond the normal range of human experience, for example, rape, torture, genocide, and war zone stress . The ranIn the US, nearly 1 in 5 adults reports taking herbal products to treat a medical illness; in fact, as recently as 1980, the US Pharmacopeia consisted of 59% herbal medicines . Green tAlthough little is known about the pharmacokinetics of L-theanine in humans, animal studies show that it is absorbed in the small intestine via sodium-coupled active transport, crosses the blood brain barrier via a leucine-preferring transport system, and is metabolized by the kidneys . In ratsIn addition to the study by Junega and colleagues, other researchers have investigated the ability of L-theanine to attenuate anxiety. Lu and colleagues compared the acute effects of L-theanine, alprazolam (a prototype anxiolytic), and a placebo on the behavioral measures of anxiety in healthy human subjects using an anticipatory anxiety model. Lu and colleagues found that L-theanine may have a relaxing effect under resting conditions, but neither L-theanine nor alprazolam had anxiolytic effects in the anticipatory anxiety model . HoweverNo studies have evaluated the potential therapeutic properties of herbal medications in PTSD or changes in gene expression. Because PTSD is categorized as an anxiety or nervous disorder, it is important to study the potential benefits of L-theanine in the treatment of PTSD and the molecular pathways responsible. The purpose of this study was to investigate if L-theanine modulates gene expression in the hippocampus and amygdala, regions of the brain that have been identified as critically important areas involved in the pathogenesis of PTSD –10. ThisEighty male Sprague-Dawley rats (Harlan Sprague-Dawley Laboratories) weighing 300–350 grams were used. They were housed in groups of three in polycarbonate “shoebox-sized” cages lined with bedding. The animals went through a 7-day adaptation period in a temperature-controlled environment  with a reverse light-dark cycle where they received 12 hours of light (6:00 PM to 06:00 AM) and 12 hours of darkness (6:00 AM to 06:00 PM). They were allowed free access to food and water. The animals were only handled for weighing, drug administration, and cleaning of cages and were naïve to all instruments. After acclimation, the investigators measured baseline body weight. Statistical analysis demonstrated equivalency between the control and PTSD-stressed groups. The use of laboratory rats in this protocol was in accordance with the NIH Guide for the Care and Use of Laboratory Animals and received approval from the Institutional Animal Care and Use Committee (IACUC) at the US Army Institute of Surgical Research, San Antonio, Texas.The animals were randomly assigned to one of eight groups by use of a computer number generator. Rats were assigned to the nonstressed groups or the 3-day restraint shock PTSD rodent model groups. There were four groups in the nonstressed rats: control vehicle (C-V), L-theanine (C-L), midazolam (C-M), and L-theanine + midazolam (C-M + L). There were also four groups in the PTSD-induced rats: control vehicle (P-V), L-theanine (P-L), midazolam (P-M), and L-theanine + midazolam (P-M + L). Each animal received a subcutaneous injection of the following: (1) control ; (2) L-theanine 10 mg/kg ; (3) midStress exposure consisted of 2-hour sessions per day of immobilization and tail-shocks for 3 consecutive days. The animals were stressed in the morning (between 08:00 AM and 12:00 PM). They were restrained in a plexiglass tube, and 40 electric shocks  were applied at varying intervals (140–180 seconds). Commercial hardware and software controlled the timing and amplitude of the shock . Rats were stressed for 3 consecutive days. The rationale for the approach was because it had been previously demonstrated that repeated stress sessions for 3 days were more effective than a single stress session in producing sustained physiological and behavioral abnormalities, such as exaggerated acoustic startle response and reduced body weight [Several laboratory studies have established and tested the inescapable tail-shock model of stress in rats. These tests have verified the neurobiological and behavioral alterations induced in the rat model and are similar to those found in human PTSD patients , 13. Ratμm thickness) in the vicinity of interaural and bregma coordinates 6.96 mm and −2.04, respectively, were dissected using a brain block (see red shaded region in μm diameter) were retrieved from the left and right amygdala . cDNA samples were assayed using a modified QIAGEN RT2 PCR array (Cat. no. PARN-60) according to manufacturer's instructions [2A receptor (Htr2a) (Cat. no. PPR06850) [1 adrenergic receptor (Adra1a) (Cat. no. PPR43329) [RNA was isolated using the QIAGEN RNEasy Mini Kit (Cat. no. 74104) following the manufacturer's protocol for tissue. RNA quality was determined using the Agilent Bioanalyzer (Agilent) with RNA 6000 Nano Kits . Total RNA yield, 260/280, and 260/230 ratios were measured using a NanoDrop spectrophotometer (Thermo). A reverse transcription reaction using 500 ng of total RNA was completed using the QIAGEN RTructions . This arructions ; 5HT2A rPR06850) ; alpha1 PR43329) –21; and PR43329) .2 profiler PCR array profiles the expression of 84 genes involved in modulating the biological processes of neurotransmitter biosynthesis, uptake, transport, and signaling through neurotransmitter receptors. This array contains receptors for specific neurotransmitters, such as acetylcholine, benzodiazepine, dopamine, gamma-aminobutyric acid (GABA), glutamate, serotonin, somatostatin, and neuropeptides. Genes involved in the regulation of neurotransmitter levels were included as well. Analysis of the expression of a focused panel of genes related to neuronal systems with this array can be performed using real-time PCR.The rat neurotransmitter receptors and regulators RTα = .05) was obtained, with the Tukey post hoc test. A variance explained statistic eta-squared (η2) was used as the reported effect size, and though ascertaining what constitutes a small/medium/large effect is primarily a function of the context/discipline, for this report .01/.059/.138 was small/medium/large [For this cross-sectional, randomized, prospective study, a one-way ANOVA was performed to compare the 8 groups, of which each unit was randomly allocated. All assumptions were examined  and in the event that the assumptions are not tenable remedial  or alternative  strategies were considered. Multiple comparison procedures (MCP) were performed in the event that significance  was chosen to serve as a springboard for discussion and interpretation of relative between-group differences on gene expression.This study was designed to investigate the effects of L-theanine on PTSD-induced changes in rat brain gene expression. Eighty-eight genes were investigated in this study. Data analysis showed a number of significant differences in gene expression in both the hippocampus and amygdala. Volcano plots were developed to compare the gene expression of the 40 nonstressed to the 40 PTSD-stressed rats for both the hippocampus and amygdala as demonstrated in P = .011, η2 = .218. There was one significant post hoc test with the P-M group having a significantly higher mean (M = 5.58) than the P-V group (M = 4.57). A significant difference was found between groups for the Maoa gene: P = .024, η2 = .194. The post hoc test found one significant pairwise comparison: (1) the C-M + L group had the highest mean (M = 3.32) and the P-V group had the lowest (M = 3.12). The Anxa9 gene was found to have significance between groups: P = .031, η2 = .187. Multiple comparison tests resulted in one significant pairwise result: the P-M group had a significantly higher mean (M = 10.73) than the P-V group (M = 10.06).In the hippocampus, the genes with an effect size > 0.138 are displayed in P = .06, η2 = .166. The P-M group had the highest mean (M = 4.2) and the P-V group had the lowest (M = 3.82). Significant between-group differences were not obtained for the Gabrb2 gene: P = .123, η2 = .142. The P-M group had the highest mean (M = 3.32) and the P-V group had the lowest (M = 2.75). Finally, the Chrm2 gene did not show significance between groups. A statistically significant difference was not obtained: P = .138, η2 = .138. Though not significant, the C-M + L group had the highest mean (M = 10.56) and the P-V group had the lowest (M = 9.96).The following genes in the hippocampus did not show significant differences between groups; however, due to the large effect size (variance explained) these genes are presented in P = .001, η2 = .271. Significant post hoc pairwise comparisons were such that the C-L group (M = 3.52) had a higher mean than each of the following: (1) P-L (M = 3.04), (2) P-M (M = 3.11), and (3) C-M group (M = 3.09). A significant difference for the Slc5a7 gene was found: P = .024, η2 = .195. The C-V group had the highest mean (M = 7.75) and the P-L group had the lowest (M = 6.67); however as per the post hoc test, this pairwise comparison was not significant. Significant between-group differences were also found for the Drd2 gene: P = .02, η2 = .189. There was a significant difference between P-V (M = 6.76) and the P-M group (M = 4.48). The Drd1a gene was also significant: P = .038, η2 = .181. A significant difference was found between the C-M + L (M = 6.41) and the P-M group (M = 4.83). Lastly, the Glra2 showed significance: P = .04, η2 = .179. There was one significant post hoc test between the C-M + L group with the higher mean (M = 5.62) and the P-M group with the lower mean (M = 4.89).Genes in the amygdala with an effect size greater than 0.138 are displayed in P = .075, η2 = .159. The C-M group had the highest mean (M = 16.51) and the C-M + L group had the lowest (M = 15.5). The Htr3a gene showed no significant difference between the groups: P = .081, η2 = .156. The C-L group had the highest mean (M = 6.74) and the P-M + L group had the lowest (M = 6.35). The Chrnb4 also showed no significant difference: P = .087, η2 = .154. The C-V group had the highest mean (M = 11.59) and the C-M + L group had the lowest (M = 10.43). No significant difference was found for the Gabrd gene between the groups: P = .089, η2 = .153. The C-M + L group had the highest mean (M = 5.23) and the P-M group had the lowest (M = 4.56). The Prima1 gene did not have a significant difference between the groups: P = .111, η2 = .145. The P-V group had the highest mean (M = 6.94) and the P-M group had the lowest (M = 6.25). No difference between the groups was found for the Chrna3 gene: P = .127, η2 = .141. The P-L group had the highest mean (M = 10.56) and the C-M + L group had the lowest (M = 9.67).Again the following genes were not found to be significant; however, based on the large effect size (variability) used in the study these genes are included in L-theanine has previously been demonstrated to produce anxiolytic effects alone or in combination with concomitant administered benzodiazepines . No studWe found a downregulation of the Egr1 gene in the PTSD-vehicle group compared to the PTSD-midazolam group. Egr1 is an early growth response nuclear transcription factor that plays a role in regulating the expression of genes required for learning and memory , 24. In Decreased Maoa enzymatic activity leads to increased concentrations of its substrate biogenic amines, including norepinephrine and serotonin. As reviewed by Bortolato and colleagues, previous research has demonstrated strong links between decreased Maoa activity and a number of reactive social behaviors, including specific links to violence and aggression . SimilarAlthough no significant difference was found between groups, the PTSD-vehicle group had the lowest mean expression of the S100a10 gene and the PTSD-midazolam group the highest. The S100a10 gene and its associated protein p11 are important in the regulation of serotonin receptors , and decA downregulation of the Gabrb2 gene in the PTSD-vehicle group compared to the PTSD-midazolam group was similar to Zhao and colleagues findings of a significantly decreased expression of the Gabrb2 gene in the anterior cingulate cortex in subjects suffering from major depressive disorder or bipolar disorder .A downregulation of the Anxa9 gene was found in the PTSD-vehicle group compared to the PTSD-midazolam group. The Anxa9 gene, part of the calcium-dependent phospholipid-binding annexin family, may play a role in suppression of apoptosis and is currently under investigation as a therapeutic target for the treatment of breast cancer . In a stAccording to Ponomarev and colleagues, downregulation of the Gabra4 gene can be correlated with stress enhanced fear learning (SEFL) in rats. These data suggest a significant role of GABA signaling in SEFL. The method of inducing SEFL was similar to the method of inducing PTSD in our study . InteresPCR analysis of tissue samples from the amygdala showed a 2.28-fold increase in Drd2 gene expression in the PTSD-vehicle group compared to the PTSD-midazolam group, which is inconsistent with the findings of a study that investigated the effects of maternal deprivation and chronic mild stress in rats . Zhu andDrd1a genes were significantly downregulated in the PTSD-midazolam group compared to the nonstressed-midazolam + L-theanine group concurrent with the results of another study exploring the effects of stress on the mouse prefrontal cortex transcriptome, demonstrating a deregulation of the Drd1a gene in chronic mild stress exposed subjects .The SLc5a7 gene was significantly downregulated in the PTSD-L-theanine group compared to the nonstressed-vehicle group. In comparison the SLc5a7 gene was found to be upregulated in tasks that required higher levels of attention, and it was found that decreased levels of cholinergic activity are involved in ADHD .Prima1 DNA methylation increased by 10–15% in subjects suffering from major depressive disorder  which inAlthough no studies have investigated gene expression changes of Chrnb4 in PTSD or anxiety, variations in this gene were shown to increase sensitivity to nicotine's effects on attention, mood, and vulnerability to tobacco dependence . The HtrRT-PCR analysis revealed significant changes between groups in several genes implicated in a variety of disorders ranging from PTSD, anxiety, mood disorders, and substance dependence. These data further elucidate the transcriptomic footprint of PTSD in the rodent amygdala and hippocampus as well as transcriptome changes effected by L-theanine and midazolam interventions.These different neurotransmitter genes are significant biologically and clinically as they are associated with various central nervous system pathologies. The hippocampal genes related to long-term potentiation (Egr1), violence and aggression (Maoa), and major depressive and bipolar disorder  were shown to be downregulated in PTSD animals and expression levels significantly changed. Other neurotransmitter genes Gabrb2 and Chrm2 were also modulated across treatment groups and warrant further exploration as they are associated with depression related clinical conditions. Amygdalar genes that were changed across treatment groups also were related to psychological conditions such as depression (Drd2), stress enhanced fear learning (Gabra4), chronic mild stress (Drd1a), and anxiety, mood disorders, and psychoses (Htr3a). These findings taken together demonstrate a relationship of possible gene expression changes in important and known neurotransmitter system genes and associated clinical pathologies. This study lays the foundation for future studies to interrogate and investigate the translation of these genes into proteins and the potential for these proteins to act as sites for pharmacologic therapeutic intervention.This understanding of the effect of PTSD at the level of mRNA transcription contributes to a more complete understanding of the overall metabonomics of PTSD and may allow for more targeted pharmacologic intervention in the future.The next step to consider in clarifying the molecular mechanism of PTSD may be to characterize PTSD-induced changes to the rat proteome. Changes in mRNA expression are often linked to changes in protein expression, but not in a predictable manner. Small changes in mRNA expression can lead to variable changes in corresponding protein expression. We propose repeating the current experiment using protein assay methods to correlate changes in mRNA expression with changes in protein expression induced by PTSD and the effects of L-theanine and midazolam on these changes. Multiple methods and computational approaches to protein assays are available .We anticipate further investigation into the network of gene relationships between control and PTSD-induced rats using bioinformatic methods such as gene enrichment analysis and network enrichment analysis. Characterization of a comprehensive proteomic network in both control and PTSD-induced rats will allow for comparative proteomic analysis that may lead to the identification of biomarkers for PTSD susceptibility as well as potential targets for pharmacologic interventions –49. Addi


Materials and Methods. 80 individuals were enrolled in this study; they were categorized into 4 groups: group 1 healthy individuals (NC) (n = 20), group 2 chronic hepatitis C virus (HCV) patients (n = 20), group 3 cirrhotic patients (LC) (n = 20), and HCC group (n = 20). Using ELISA technique serum levels of IL-6, IL-10, and alpha fetoprotein (AFP) were evaluated in all groups. Results. The mean serum levels of IL-6 were significantly higher in HCC than in LC, HCV, and NC groups , respectively (P < 0.05); also the serum levels of IL-10 were significantly higher in HCC compared with LC, HCV, and NC groups  (P < 0.05). We also found that the tumor size is correlated strongly with IL-6 and IL-10 levels , respectively. Conclusion. The combination of those markers may help to identify a group of HCC patients with low AFP.Interleukin-10 (IL-10) and interleukin-6 (IL-6) have been reported to be related to hepatocellular carcinoma (HCC) prognosis. This study aimed to investigate the clinical usefulness of serum levels of IL-6 and IL-10 as biomarkers for HCC among high-risk patients. Hepatocellular carcinoma is one of the most common malignant tumors, representing more than 5% of all cancers. The estimated annual number of cases exceeds 500,000, with a mean annual incidence of around 3-4%. In terms of relative frequencies, HCC ranks as the fifth most common cancer in the world; it is also the fifth among men and eighth among women; it is the second among cancers of the digestive tract after stomach cancer . The main risk factors for HCC are hepatitis B virus (HBV), HCV, alcohol, aflatoxin, and possibly obesity and diabetes. Together, HBV and HCV account for 80% to 90% of all cases of HCC worldwide , 3. Egypt has possibly the highest HCV prevalence in the world; 10%–20% of the general population is infected and HCV is the leading cause of HCC and chronic liver disease in the country. Approximately 90% of Egyptian HCV isolates belong to a single subtype, 4a, which responds less successfully to interferon therapy than other subtypes . The burThe single most important tumor marker for HCC is AFP. HCC surveillance with serum AFP level and ultrasonography has been recommended for patients with cirrhosis. Although the detection of serum AFP level is well established in the screening and diagnostic purpose for HCC, a major shortcoming is that serum AFP is insensitive for the early cancer detection , 8. In a prospective study by Marrero , they shGiven improvement in the overall survival of patients with cirrhosis and the continued rise in the incidence of HCC in Egypt, strategies for the detection of early HCC in the at-risk population may lead to improved survival of patients with this deadly tumor . The poor sensitivity of AFP renders it unsatisfactory for this purpose and suggests the need for novel biomarkers for the detection of early HCC . Several Mounting evidence indicates the involvement of cytokines in hepatocarcinogenesis , 13. IL- IL-10 is a pleiotropic cytokine produced by macrophages, T-helper 2 (Th2) cells and B-lymphocytes, and both can stimulate and suppress the immune response. IL-10 has been shown to inhibit various immune reactions . Recently, it has been proposed that IL-10 plays a key role in the oncogenetic and metastatic ability of neoplasms . IncreasSerum IL-10 concentration has been reported to be significantly elevated in patients with chronic HCV and IL-10 may be related to hepatocarcinogenesis with suppression of immune surveillance . Hsia et al.  reportedThe aim of this study is to investigate and analyze the discriminate power of serum IL-6 and IL-10 individually and whether their combination with AFP would increase accuracy in discriminating Egyptian patients with HCC from healthy and cirrhotic subjects. Up to our knowledge a similar study was not done before in Egypt.Eighty individuals were included in this study. They were selected from Tropical and Internal Medicine Department in a big medical center in Cairo.This study comprises the following groups. Group 1. Normal control (NC) group, including 20 apparently healthy volunteers . They are negative for HCV-Ab and have normal abdominal ultrasonography with no evidence of liver disease and/or of neoplasm. Group 2. HCV group, including 20 patients . They are infected with HCV (positive for both HCV-Ab and HCV RT-PCR). Group 3. Cirrhotic (LC) group, including 20 patients . They are suffering from liver cirrhosis with no histological evidence of cancer. Group 4. HCC group, including 20 patients . HCC was confirmed by pathology, imaging (computer tomography (CT) and ultrasound), and serum AFP. Tumor size was measured according to CT imaging.All the studied groups included in this study were free from heart diseases, kidney diseases, muscle disorders, pancreatitis and bilharziasis, and HBV. Also patients with special habits like smokers and alcoholics were excluded from this study to prevent any external interference.All patients and controls gave their informed consent which was ethically conducted in accordance with the Helsinki Declaration. Ten milliliters of fasted venous blood were taken from patients and controls; serum was separated by centrifugation and stored at −80°C until further examinations.Clinical assessment: all the patients were subjected to abdominal ultrasound, liver biopsy, and laboratory assessment.Laboratory evaluation including hepatitis C virus: anti-HCV antibodies by third generation ELISA   and RT-Phepatitis B virus: HbsAg and anti-HBc were performed by a direct noncompetitive sandwich assay  based on ELISA technique . IL-10 and IL-6 and AFP were detected using a third generation ELISA  . Liver function tests: A diazotization method used for determination of serum total bilirubin . ActivitBlood picture was done on Coulter Counter T890,  . Histological studies: HCC neoplastic cells were identified histopathologically in H&E-stained sections of a core needle biopsy. All patients were subjected to the following. All tests were done according to the manufacturer's instructions.P value < 0.05 was considered significant [Data were analyzed by SPSS statistical package version 19. One-way ANOVA, Duncan multiple comparisons, and Pearson's correlation coefficient were used for analysis. Multiple receiver operating characteristic curves (ROC curves) were drawn to assess the validity of the tumor markers. A nificant .P = 0.001). The serum levels of AFP were significantly elevated in HCC group compared with both HCV group and LC group (P = 0.01); there was also a significant increase in AFP level in LC group as compared with HCV group (P < 0.05). As shown in P = 0.001), and there is a consistent increase in the IL-6 level with the disease progression from NC to HCC. The serum levels of IL-6 were significantly elevated in HCC group compared with both HCV group and LC group (P < 0.05) but no significant difference in IL-6 level between HCV group and LC group.The serum levels of IL-6 were significantly higher in HCC, LC, and HCV groups  than NC group (2.57 ± 0.31) (P = 0.001). The serum levels of IL-10 were significantly elevated in HCC group compared with both HCV and LC groups (P < 0.05); however, there was no significant difference in IL-10 level between HCV group and LC group. The serum levels of IL-10 were significantly higher in HCC, LC, and HCV groups  than NC group (3.31 ± 0.42) , respectively. In HCC patients tumor size was correlated strongly with IL-6 and IL-10 levels , respectively . But there was no type of correlations between serum IL-6 or IL-10 and serum AFP in HCC and LC groups.In HCV group there was a significant correlation between AFP and IL-6 (Receiver operating characteristic (ROC) curves were plotted to define the optimal cutoff values and to identify the specificity and sensitivity for serum AFP, IL-6, and IL-10 in differentiating HCC from benign liver diseases  .At serum level 20 ng/mL , the AUROC for AFP was 0.76 (95% CI: 56.3–94.1), with a sensitivity of 80%, specificity 80%, and accuracy 80%.  The AUROC for IL-6 was 0.931 (95% CI: 0.851 to 0.975), with a sensitivity of 90%, specificity 86.67%, accuracy 87.5%, and optimal cutoff value 8.6 pg/mL . The AURThe values for the combined detection using more than one marker are also presented in HCC is responsible for a large proportion of cancer deaths worldwide. HCC is frequently diagnosed after the development of clinical deterioration at which time survival is measured in months. Long-term survival requires detection of small tumors, often present in asymptomatic individuals, which may be more amenable to invasive therapeutic options. Surveillance of high-risk individuals for HCC is commonly performed using the serum marker AFP often in combination with ultrasonography. Serum AFP used alone can be helpful if levels are markedly elevated, which occurs in fewer than half of cases at time of diagnosis , 30. This study revealed a significant increase in AFP serum levels in HCC and some benign liver diseases such as hepatitis and cirrhosis. AFP was significantly lower in benign liver diseases as compared with HCC. Similar results were obtained by many studies –32. In t In the present study we found that 20% (4/20) of HCC patients had serum AFP level <20 ng/mL. The diagnosis of HCC could be very difficult in high-risk patients when these patients had low AFP level or tumors mimicking HCC. It has been estimated that up to 75% of patients with small HCC and 20% of patients with large HCC may have normal serum AFP level that could escape from HCC surveillance , 38; how Both IL-6 and IL-10 are multifunctional cytokines produced by a range of cells and play a central role in host defense mechanism and modulation of immune response . An incrThe results of the present study showed that serum levels of IL-6 were significantly higher in all patients groups compared with control group, and we found significantly higher circulating IL-6 titers in HCC than in both cirrhotics and HCV groups. Our findings were in agreement with the results of other studies, Porta et al.  Hsia et Our results were not consistent with Zekri et al.  and ToveThere are many reasons to make IL-6 as intriguing cytokine to study in HCC patients. Naugler and Karin  and Jin IL-6 was also shown to induce the expression of the mitogenic, morphogenic, and proneoangiogenic scatter factor hepatocyte growth factor which, besides being commonly expressed at high levels in HCCs, also signals using the same STAT-3 pathway as IL-6 , 50.Porta et al.  reportedIL-6 may also decrease HCC cell apoptosis, thus conferring a survival advantage to the tumor; indeed, in a mouse model, IL-6 proved to reduce Fas-induced apoptosis . IL-6 maIL-6 has also been proposed as a cause of natural killer cell dysfunctions, thus potentially representing a mechanism of tumor escape from immune surveillance .In this study we found an elevated level of circulating IL-10 in patients with HCV, cirrhosis, and HCC and the concentrations are associated with disease progression.These results were in agreement with Hattori et al. , who fouIL-10 cytokine is an anti-inflammatory endogenous mediator through the inhibition of the production of proinflammatory cytokines. Zekri et al.  reported The immunosuppressive effects of IL-10 may play a major role in the development of neoplastic processes by suppressing macrophage activation and interferon-gamma production, thereby crippling two potential mediators of an antitumor response; this may help the tumor cells escape host immune surveillance and potentiate tumor cells to metastasize. The functional consequences of IL-10 binding to its receptors on tumor cells could be the prevention of programmed cell death and the promotion of proliferation. In addition, IL-10 may contribute to the development of an environment favorable to neoplastic cells and to the enhancement of their metastatic potential . In the current study, the sensitivity, specificity, and accuracy of AFP, IL-6, and IL-10 were 80%, 80%, and 80%; 90%, 86.67%, and 87.5%; 80%, 96.67%, and 92.5%, respectively. Using the optimal cutoffs derived from the ROC curves IL-6 level was elevated in 3 patients out of 4 who were with AFP <20 ng/mL, while IL-10 level was elevated in 2 of them. Our results indicated a potential role for IL-6, and IL-10 as a tumor marker for HCC with respect to AFP and this was in agreement with the previous studies , 32, 37. Using the optimal cutoffs derived from the ROC curves a combination of IL-10, IL-6, and AFP produced better sensitivity (92%) and specificity (92.3%), so discriminating analysis based on AFP, IL-6, and IL-10 had high diagnostic accuracy in discriminating HCC from non-HCC benign liver diseases, especially among patients with low (<20 ng/mL) AFP level, and these results were in agreement with the previous studies , 55, 56.Another interesting finding in our study is that IL-6 and IL-10 values were correlated significantly with the tumor size which is consistent with results obtained by Malaguarnera et al. .In our study there was a strong correlation between serum IL-6 and IL-10 levels in patients with LC and HCC and this agreed with Gastl et al. .High levels of IL-6 and IL-10 are observed in HCC patients, correlate with tumor size, and may be helpful to identify a subset of HCC patients with low AFP level. Serum IL-6 and IL-10 levels may serve as complementary tumor markers and contribute to the differential diagnosis in HCC patients.